Brain Regional Differences in Hexanucleotide Repeat Length in X-Linked Dystonia-Parkinsonism Using Nanopore Sequencing

Reyes, Charles Jourdan; Laabs, Björn‐Hergen; Schaake, Susen; Lüth, Theresa; Ardicoglu, Raphaela; Raković, Aleksandar; Grütz, Karen; Alvarez‐Fischer, Daniel; Jamora, Roland Dominic G.; Rosales, Raymond L.; Weyers, Imke; König, Inke R.; Brüggemann, Norbert; Klein, Christine; Dobričić, Valerija; Westenberger, Ana; Trinh, Joanne

doi:10.1212/nxg.0000000000000608

Cited by 21 publications

(23 citation statements)

References 25 publications

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“…DNA was extracted with the Blood and Cell Culture DNA Midi kit (Qiagen). Long-range PCR was performed to amplify the TAF1 SVA (amplicon Size: 3.2 kb) in XDP patients, as previously described [ 7 ], using the PrimeSTAR GXL DNA Polymerase ® (Takara Bio). The primer sequences are documented in Supplementary Table S2 .…”

Section: Methodsmentioning

confidence: 99%

“…The repeat number is inversely correlated with age at onset and disease severity [ 5 , 6 ]. In addition, somatic mosaicism has been observed, with a higher median number of repeats detected in the cerebellum and basal ganglia compared to blood [ 7 ]. In XDP patients, seven variants have been found on the X chromosome: five single-nucleotide variants (SNVs), a 48-bp deletion and the SVA insertion [ 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

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Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing

Lüth

Laβ

Schaake

et al. 2022

Genes

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Background: X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. Methods: Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach. Results: High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus. Conclusions: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing

Lüth

Laβ

Schaake

et al. 2022

Genes

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“…All DNA was extracted from the Blood and Cell Culture DNA Midi kit (Qiagen). Sequencing was performed for the SVA amplicon (3.2kb) and Cas9 enrichment as previously described 8 , with super accuracy model for base calling and mapped to reference (detailed in Supplementary figure 1 ). Long- range PCR was performed for the SVA (3.2kb) as previously described1.…”

Section: Methodsmentioning

confidence: 99%

“…Mosaic modifiers, which exist in every patient at variable frequencies, have not been studied extensively. We previously described the presence of somatic mosaicism in XDP with a higher number of repeats detected in the cerebellum and basal ganglia compared to blood 8 . In this present study, we identified novel mosaic repeat motif patterns that deviate from the known hexanucleotide repeat motif and investigate whether they act as new genetic modifiers of repeat instability and AAO of this neurodegenerative disorder.…”

Section: Introductionmentioning

confidence: 99%

Mosaic deletions in X-linked dystonia-parkinsonism influence repeat stability and disease onset

Trinh

Lueth

Schaake

et al. 2022

Preprint

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Background: While multiple genetic causes of movement disorders have been identified in the past decade, modifying factors of disease expression are still largely unknown for most conditions. X-linked dystonia-parkinsonism (XDP) is an inherited neurodegenerative disease caused by a SINE-VNTR-Alu (SVA)-type retrotransposon insertion that contains a hexanucleotide repeat within an intron of the TAF1 gene. To date, four putative genetic modifiers explain about 65% of variance in age at onset in XDP. However, additional genetic modifiers are conceivably at play in XDP and may include mismatches of the SVA hexanucleotide repeat motif. We aim to identify additional genetic modifiers of XDP expressivity and age at onset (AAO). Methods: Third-generation sequencing of PCR amplicons from XDP patients (n=202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment were used to confirm the presence of identified repeat mismatches. Results: An increased frequency of deletions at the beginning of the hexanucleotide repeat (CCCTCT)n domain was found. Specifically, three deletions at positions 11, 14, and 17 of the TAF1 SVA repeat motif of somatic mosaic origins were detected in different combinations. The most common one was three deletions (1-2-3) at a median frequency 0.425 (IQR:0.42-0.43) and deletions within positions 11 and 14 (1-2-wt) at a median frequency 0.128 (IQR:0.12-0.13). The frequency of deletions at positions 11 and 14 correlated with repeat number (r=-0.48, p=9.5x10-13) and AAO (r=0.34, p=9.5x10-7). The association with AAO still stands when including other modifier genotypes (MSH3 and PMS2) in a regression model. However, the association dissipates when including repeat numbers. Conclusion: We present a novel mosaic repeat motif deletion within the hexanucleotide repeat (CCCTCT)n domain of TAF1 SVA. Our study illustrates: 1) the importance of somatic mosaic genotypes; 2) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; 3) that these variations may remain undetected without assessment of single molecules.

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“…Control tissues included PFC ( n = 3), MC ( n = 1), CB ( n = 2), AP ( n = 1), caudate nuclei (CN, n = 3) and thalamus (TH, n = 1). Hexamer repeat length for the processed XDP samples was genotyped by Fernandez-Cerado et al 21 and Reyes et al 22 and repeat numbers are as follows: 17.01, 44; 17.06, 41; 17.10, 34; 17.12, 42; 17.13, 46; 17.17, 54; L-7.995, 45; L-10.332, 41.…”

Section: Methodsmentioning

confidence: 99%

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism

Capponi

Stöffler

Penney

et al. 2021

Brain Communications

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X-linked dystonia-parkinsonism (XDP) is a monogenic neurodegenerative disorder of the basal ganglia, which presents as a combination of hyperkinetic movements and parkinsonian features. The underlying genetic mechanism involves the insertion of a SINE-VNTR-Alu retrotransposon within the TAF1 gene. Interestingly, alterations of TAF1 have been involved in multiple neurological diseases. In XDP, the SINE-VNTR-Alu insertion in TAF1 has been proposed to result in alternative splicing defects, including the decreased incorporation of a neuron-specific microexon annotated as 34′. This mechanism has become controversial as recent studies failed to provide support. In order to resolve this conundrum, we examined the alternative splicing patterns of TAF1 mRNAs in XDP and control brains. The impact of the disease-associated SINE-VNTR-Alu on alternative splicing of microexon 34′ was further investigated in cellular assays. Subsequently, microexon 34′ incorporation was explored by RT-PCR and Nanopore long-read sequencing of TAF1 mRNAs from XDP and control brains tissues. Using cell-based splicing assays, we demonstrate that presence of the disease-associated SINE-VNTR-Alu does not affect the inclusion of microexon 34′. In addition, we show that (1) microexon 34′-containing TAF1 mRNAs are detected at similar levels in XDP as in controls and that (2) the architecture of TAF1 transcripts is remarkably similar between XDP and controls brains. These results indicate that microexon 34′ incorporation into TAF1 mRNA is not affected in XDP brains. Our findings shift the current paradigm of XDP by discounting alternative splicing of TAF1 microexon 34′ as the molecular basis for this disease.

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Brain Regional Differences in Hexanucleotide Repeat Length in X-Linked Dystonia-Parkinsonism Using Nanopore Sequencing

Cited by 21 publications

References 25 publications

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing

Mosaic deletions in X-linked dystonia-parkinsonism influence repeat stability and disease onset

Dissection of TAF1 neuronal splicing and implications for neurodegeneration in X-linked dystonia-parkinsonism

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